Process for a microbial polysaccharide



Jan. 11, 1966 M. c. CADMUS ETAL 3,228,855

PROCESS FOR A MICROBIAL POLYSACCHARIDE Filed Oct. 24, 1963 4 Sheets-Sheet 1 0 9 g 8,000 g, E V E .5 a, 0 0 2 a 6,000 g C :03 3 '5 a", 4,000 5 a Q.

0 0 0 0| 0.2 0.3 0.4 0.5 0.6 0.7 0.8 Enzyme-hydrolyzed casein (g/l00 ml) FIGURE l INVENTORS MARTIN C. CADMUS BY l/,1 ET AL ATTORNEYS Jan. 11, 1966 M. c. CADMUS ETAL 3,228,855

PROCESS FOR A MIGROBIAL POLYSACGHARIDE Filed Oct. 24, 1965 4 Sheets-Sheet 5 2% KCl; 70 I2| C/l5 min.

no KCI; a? no heat 8 30 ,v a f '5 2/ KCI; 20 no heat I I ..-V I0 no KCI no heat O 0.5 L0 L5 Polymer concentration (g/IOO ml) FIGURE 3 INVENTORJ MARTIN c. CADMUS ET AL ATTORNEYS Jan. 11, 1966 M. c. CADMUS ETAL 3,228,855

PROCESS FOR A MICROBIAL POLYSACCHARIDE Filed Oct. 24, 1963 4 Sheets-Sheet 4 NuCl COCl

IO Salt concentration (g/IOO ml) Viscosity (centipoise x IO' INVENTORJ MARTIN C. CADMUS BY fl ET AL ATTORNEY 5 United States Patent "ice 3,223,855 PRQCESS FOR A MICROBIAL POLYSACCHARIDE Martin C. Cadmus and Ralph F. Anderson, Peoria, 111., assignors to the United States of America as represented by the Secretary of Agriculture Filed Oct. 24, 1963, Ser. No, 313.9769 1 laim- (CL 1 -3 (Granted under Title 35, U.S. Code (1952), sec. 266) A nonexclusive, irrevocable, royalty-free license in the invention herein described, throughout the world for all purposes of the United States Government, with the power to grant sublicenses for such purposes, is hereby granted to the Government of the United States of America.

This invention relates to a novel microbial polysaccharide, aqueous solutions of which novel polysaccharide polymer display unique viscosity characteristics that make the edible polymer particularly advantageous as a thickening agent broadly and as an additive for oil well drilling fluids and foodstuffs in particular. This invention also relates to a process for obtaining the novel polysaccharide in yields of 40-45 percent based on the carbohydrate nutrient rather than in commercially unattractive yields of only about 15-20 percent obtained when the amount of nitrogen source in the fermentation medium is not restricted (see FIG. I) and certain metal ions, namely Mg and Mn are not available.

More particularly this invention relates to the production in commercially acceptable yields of a novel watersoluble uronic acid polysaccharide, the constituent sugars of which are glucose and galactose, by fermenting heretofore unclassified microorganism belonging to the genus Arthrobacter, no prior art members of which genus have ever been known to produce a polysaccharide gum.

Still more particularly this invention relates to the discovery that certain previously undescribed microorganisms belonging to the diphtheroidic genus Arthrobacter are capable of producing high yields of a novel water-soluble polysaccharide when fermented under certain aerobic conditions for 3 to 4 days in a medium comprising carbon and restricted nitrogen sources, as well as, preferably, trace amounts of manganese in addition to obligatory amounts of magnesium ion.

Certain microbial polysaccharide gums are widely known, e.g., the dextrans which consist entirely of glucose units and are produced from sucrose by the enzymes of Leuconstoc bacteria. Less well known are the phosphomannan gums produced by yeasts belonging to the genus Hansenula, Anderson et al., Arch. Biochem. Biophys. 89: 289 (1960) whose constituent sugars are mannose and mannosee6-phosphate, the polysaccharide produced by Cryptococcus laurenlii, containing xylose, mannose, glucuronic acid, and acetyl in the ratio of l:4:1:1, Cadmus et al., Appl. Microbiol., 10: 1953 (1962), as well as polysaccharide B-1459 produced by the bacterium Xanthomonas campestris, NRRL 13-1459, Rogovin et al., J. Biochem. Microbiol. Technol. Eng. 3: 51 (1961) which consists of mannose, glucose, glucuronic acid and acetyl in the ratio of 2: l 1 1.

As already indicated, the constituent sugars of the polysaccharide produced by Arthrobacter viscosus NRRL B- 1973 and NRRL 3-1797 are D-glucose and D-galactose in equimolar proportions, there also being an equimolar proportion of D-mannuronic acid moieties and 5 molar equivalents of O-acetyl groups linked to 5 of the 8 nominal hydroxyl positions of the sugars, thus constituting 25.5 percent of the dry Weight of the polysaccharide, see Slonekar et al., p. 24D, Abstr. of Papers, 145th Meeting of A.C.S., New York City, September 9, 1963. In other words the B-1973 polysaccharide comprises highly acetylated repeating units consisting of a mannopyranosyluronic 3,223,855 Patented Jan. 11, 1966 acid residue linked beta 1:3 to a glucopyranosyl residue linked beta 1:4 to a galactopyranosyl residue which latter is again linked 1:4 to a beginning mannopyranosyluronic acid residue.

One object of our invention is the production of a novel polysaccharide having unique and advantageous properties in solution. Another object is a process for producing a polysaccharide gum polymer by fermenting members of a novel species of diphtheroidic bacterium found in a soil sample from Guatemala and now definitively identified as belonging to the genus Arthrobacter.

In accordance with standard taxonomic and physiological findings, herein set forth, we have classified the novel polysaccharide-producing organism as belonging to the genus Arthrobacter and have assigned them to the species name visco-sus as weil as the accession numbers NRRL 13-1973 and NRRL 13-1797.

In accordance with the objects of our invention we have made the discovery that two virtually indistinguishable strains of Arthrobacter, specifically A. vi'scosus NRRL B-l9,73 and A. viscosus NRRL 13-1797 under certain fermentation conditions elaborate excellent yields of an edible polysaccharide gum which has a novel constitution and unique characteristics and properties.

Parallel fermentations with inocula of the above organisms were made in media comprising enzyme-hydrolyzed casein equivalent to 0.04 percent total nitrogen as the nitrogen source and respectively dextrose, technical maltose, or pure maltose as the carbon source. Because the yields with technical maltose were 2 to 3 times that obtained with the other carbohydrates, we sought an explanation. Addition to the dextrose-containing medium of the two amino acids known to be present in crude maltose gave no improvement in yield. However, the addition of ash from a sample of technical maltose increased the yield of the polysaccharide by two to three fold, and it was subsequently determined that magnesium was mainly responsible, a level of 0.08 percent MgSO, 71-1 0 being optimal, traces of the manganese ion also being required, Polysaccharide production was found to be unaffected by the presence or absence of ferric, cobalt, or molybdenum ions. Maximum yields of polysaccharide having a maximum molecular weight are obtained when the pre-sterilization pH of the medium is adjusted to pH 7.0 and 0.4 percent of KH PO butter is added. When 0.& percent buffer was used the yield was very slightly greater but the polymer was more crystalline in nature and probably had a lower molecular weight inasmuch as the viscosities obtained in solution were lower.

The distinguishing characteristics of our novel polysaccharide polymer are exemplified in the viscosity responses of dilute solutions thereof to heat (FIG. 11) and to salts, especially aluminum sulfate, shown in FIGS. III and IV.

Whereas the viscosities of solutions of Other polysaccharides decrease, particularly in the presence of a salt, as the temperature is increased, solutions of our novel polysaccharide actually increase in viscosity as the tem perature is increased, and on cooling the solution, the viscosity then increase further. On autoclaving at 121 C. for 1 5 minutes and cooling, solutions containing about 1.5 percent or more of the polysaccharide actually be- CQme gels that tend to retain the shape of the container. In the presence of KCl, a 0.75 percent solution of the polysaccharide gels on cooling. The addition of 2 percent KC-l, even without heating to solutions containing 0,5 percent or more, of the polysaccharide very markedly increases the viscosity. The addition of 0.5 percent of NaCl or OaCl doubles the viscosity, but larger additions of these salts do not result in greater increases. The addition of 0.05 percent aluminum sulfate to a 1.0 percent solution of the polysaccharide is unique in that it causes a 20-fold increase in viscosity, but further additions of aluminum sulfate to beyond the 0.25 percent level result in a precipitous dropping off of viscosity to a level somewhat below that of the original solution. The viscosity is stable to pH variations within the range 5.0-10.0. It is apparent that the above responsesto temperature and to salts make the polymer very valuable for a diversity of industrial uses, e.g., as an additive for specialty meats, pudding mixes, etc. and as an important constituent of salt-resistant oil well drilling fluids.

In connection with the following specific example, it should be pointed out that the only difference between fermenting an inoculum of NRRL B1973 and of NRRL B-l797 is that the former gives a very slightly (ca. 3 per. cent) greater yield of polymer. Also, shake flasks are not suitable for obtaining suitable yields of the polysaccharide, probably'because of poor aeration after the culture fluid begins to thicken. Twenty-liter fermentors having paddles are suitable, the paddles being operated at 200 r.p.m. the first day, 300 r.p.m. the second day, and 500 r.p.m. the third and fourth days. The yields were significantly reduced when fermentations were started at 500 r.p.m.

- EXAMPLE 1 A paddled 20 l. fermentor containing 1. of a sterilized fermentation medium having a pH of 7.0 and the following composition per 100 ml.

Grams Commercial corn sugar 3 Enzyme/hydrolyzed casein 0.25

M11804 KH PO 0.4

Water q.s.a.d. 100.

was inoculated under sterile conditions with 500 m1. of material from a proliferating l-day fermentation of Arthrobacter viscosus NRRL B-1973 in the same medium and incubated at 25 C. under a positive pressure of 1 atmosphere with air admitted at the rate of 0.25 vol. air/liter per min. The paddles were operated at 200 r.p.m. during the first 24 hours, at 300 r.p.m. during the second 24 hours, and at 500 r.p.m. during the next 48 hours. The viscous culture liquor was then diluted with 4 parts of water plus /2 volume of absolute methanol and supercentrifuged for 30 minutes to precipitate cells and debris. The supernatant containing the polysaccharide polymer was then treated by adding 1 g. potassium acetate per 100 ml. (a like amount of KCl can be substituted for the acetate) and 2.5 vols. of 95 percent ethanol to precipitate 92-96 percent of the polymer therefrom.

The precipitate was collected, redissolved in water, reprecipitated, and lyophilized to obtain 1.4 g. of a dry spongy, white product from each 100 ml. of fermented liquor. Based on the initial carbohydrate nutrient, the yield was 47 percent of a polysaccharide composed of glucose, galactose, manuronic acid, and acetyl groups, which groups are present in molar proportions of 1:1:1:5.

Tables I and II summarize the taxonomic and physiological characteristics of Arthrobacter viscosus NRRL B-1973 and NRRL B-1797 as compared with the corresponding spectra for known Arthrobacter species.

Strains NRRL B-l797 and B-1973 produced extremely viscous growth on a variety of media supplemented with various carbon sources. Stained cells from an 18-hour culture on yeast-malt agar showed the presence of highly pleomorphic, gram-negative cells. These cells showed definite changes in morphology from short branched, curved and straight rods at 8-12 hours to staphyococcuse like forms at 24-48 hours. Many of these cells exhibited uneven staining reaction. The cultures failed to initiate growth in synthetic medium with phenol as a carbon tics eliminate Agrobacterium radiobacter as a generic possibility.

The occasional formation of Y, T, and U forms with uneven staining appeared typical of one of the soil diphtheroids. The growth on inorganic nitrogen and the gram-negative staining reaction eliminated Corynebacterium and pointed toward Arthrobacter as the probable genus.

The isolated cultures were compared physiologically and morphologically with known species of Arthrobacter. All control cultures were grown in media in which a maximum extent of polymer production was shown by B-1973 and B- 1797. This medium consisted of 0.25 percent enzyme hydrolyzed casein, 3 percent commercial corn sugar, MgSO -7H O, MnSO '4I-I O and dibasic potassium phosphate buffer, pH 7.0. None of the control organisms produced any polysaccharide under these conditions. The differences and similarities between controls and unknown cultures are summarized in Table I.

The ditferences noted are suflicient to designate a new species. The name Arthrobacter viscosus, sp. n. is proposed with respect to the formation of slimes in simple basal medium supplemented with each of ten different carbon sources. Strain NRRL B-1797 is designated as the type species since it was the first one studied. NRRL Bl973, since it closely resembles B-1797 in physiology and morphology, is considered a strain of the same species. The description of type species NRRL B1797 follows.

Colonial Characteristics Agar streak: filiform, moderate growth, glistening, cream-white mucoid. Agar colonies: large (3 mm.) circular, smooth, entire, flat, opaque, glistening, very mucoid. Gelatin colonies: circular, raised, entire, opaque, cream, smooth, glistening, very mucoid. Soil extract agar slant: growth abundant, white, filiform, very glistening, mucoid. Asparagine agar slant: growth moderately aboundant, white, filiform, glistening, very mucoid. Asparagine colonies: 11.5 mm., circular, translucent, white. Potato: growth moderate, viscous, glistening, filiform, light brown. ll lannitol-Ca-glycerophosphate agar slant: very mucoid, white, raised, filiform, glistening' Mannitol-Ca-glycero phosphate agar colonies: 4.5 mm., spreading, raised, glistening, very mucoid, white precipitate formed around colonies, no brown precipitate. Yeast-malt slant: (Haynes et al., (1955)): heavy, mucoid, whitegrowth which is moderate in 24 hours and profuse in 48 hours.

.Physiologyz Aerobic, very slightly catalase positive.

. No acid produced from glucose, sucrose, lactose or manniplemented with various carbon sources.

tol; final pH was 7.7. Slight acid production was noted in glycerol (pH 6.2). No acid production from arabinose (pH 7.9), or-from raflinose and Z-methyl-glucoside (pH 8.3). No gas produced from any of the tested carbohydrates. No gelatin liquefaction noted after 3 months incubation. Indole not produced. Acetylmethylcarbinol not produced. Nitrite produced from'nitrates. No starch hydrolysis occurred. Litmus milk half cleared in one month; serum clear; partial reduction after one month; hard pellicle on milk after one month; no curd formed and pH of reaction slightly alkaline. Urease not 131'07 duced. Citrate utilized aslthe sole source of carbon. Hydrogen sulfide not produced from cysteine or thiosulfate. Growth occurred on inorganic nitrogen sources sup No cellulase activity. Optimum temperature, 25 C. to 28 C. Growthgreatly reduced at 37 C. as well as at 10 C. The

5 optimum pH (FIG. I) was 6.11 and growth was sharply reduced at pH values below 5.0 and above 7.0.

Strain NRRL B-1797. The optimum temperature for production of the gum was 25 C. Optimum pH for gum production was 7.0 with none produced at pH values below 4.0 with reduced production at 5.0, 6.0, 8.0, and 9.0 and none at pH 10.0 (FIG. 2).

Morphology: Cells used for study were grown in nutrient broth on a rotary shaker at 24 C.

At 12 hours, gram-negative long and short rods were common. Many T, Y, X, and U shaped cells were present as well as curved rods. Most of these cells were unevenly stained. Some of the longer rods had 2-4 heavily stained granules in a lighter stained cell. The shorter rods almost approached coccoid forms and had heavier stained ends and lighter stained center portions. At 24-48 hours staphlococcus-like groupings of cells were predominant. A few large single coccoids were present.

Flagella stains were made on cells grown on YM slants and suspended in distilled H O. Observations were made at 12 hours. Flagella were not observed, but highly plomorphic Y, T, and U forms were again observed with this staining procedure.

Strain NRRL B-1973 produced viscous growth in liquid media, but its growth on yeast-malt slants and other solid media was not extremely mucoid. Since this organism diifers in this respect and in one physiological aspect, the production of urease, it will be considered a variant of 13-1797. Following is the description of this culture.

Colonial Characteristics Agar streak: filiform, raised, glistening, viscous. Agar colonies: 3 mm., circular, raised, glistening, viscous, white opaque. Soil extract agar: filiform, moderate growth, glistening, soft, viscous, raised, cream-colored. Gelatin colonies: circular, 2-3 mm., smooth, raised, glistening, opaque, no liquefaction. Asparagine agar slant: growth moderately abundant, light cream, slightly mucoid, glistening, wrinkled edge. Asparginc agar colonies: 1

.mm., circular, translucent, white, raised, slightly mucoicl.

Potato: growth moderately abundant, glistening, soft, filiforrn, cream-buff. Yeast-malt slant: viscous, profuse white growth, slightly mucoid in 24 hours. Mannitol-Caglycerol-phosphate colonies: 3-3.5 mm., raised glistening, mucoid, circular, white precipitate formed around colonies, no browning of medium.

Physiology: Aerobic, weakly catalase positive. No acid production in glucose, sucrose, lactose, or mannitol. Pi- 1131 pH of fermentation of glucose was 7.7, sucrose and lactose, 7.5, and mannitol, 7.3. Slight acid production was noted in glycerol (pH 6.2). Basic products resulted from fermentations of arabinose, raffinose and a-methyl glucoside (pH 8.3-8.4). No gelatin liquefaction shown after 3 months incubation. Indole not produced. Acetylmethylcarbinol not produced. Nitrites were produced from nitrates. No starch hydrolysis. Litmus milk completely cleared in one month with no curd formation; reduction began in 25 days and was complete in 2 days; a hard pellicle was formed on milk in about 21 days and the reaction was slightly basic. Urease produced. Citrate utilized as the sole source of carbon. H 3 not produced from either cysteine or thiosulfate. Growth occurred on inorganic nitrogen, but only slight polymer production noted on inorganic nitrogen plus glucose; moderate polymer production shown on inorganic nitrogen plus crude maltose. Optimum temperature, 25-28 C. No growth occurred at 37 C. and little growth was shown after 3-4 days incubation at 10 C. The optimum pH (FIG. 1) was 6.1 and growth was greatly reduced at pH values below 5.0 and above 7.0.

Morphology: At 12-19 hours, short gram-negative rods were present. Some were evenly stained, but the majority were unevenly stained. Some Y, X, U, and T forms were present and these also were unevenly stained. The short rods had heavily stained ends and were lightly stained in the central portion. The longer rods appeared to have 3-6 heavy staining granules in a lighter stained cell.

After 24-48 hours micrococcus-like cell groupings were present and predominant. These cells were unevenly stained but appeared to be gram-negative.

Flagella staining of cells grown on YM slant and suspended in distilled H O, showed the presence of no flagella, but the highly pleomorphic Y, X. and U forms were plainly visible with this staining technique.

TABLE I.COUPARISONS BETWEEN KNOWN STRAINS OF ARTHROBACTER AND NEW STRAINS A. globiformis A. pascens A. pasccns A. simpler A. otrydans A. aurescens A. tumescens NRRL 13-2880 NRRL 1814 NRRL 13-2884 Jensen (1954) Sguros (1955) NRRL 13-28179 NRRL 15-2881 Gelatin liquefaction +stratilorm. Starch hydrolysis HZS production Slight in in cysteine. cysteine cysteine. thi thio Chromogenesis cream white white cream yellow and Pale yellow gray, cream gray.

Cotalase Motility NH NO utilized as sole N source.

13 thiamin, or terregens factor no (Lochhead 1953) needed for growth.

A. citrcus A. tcrregcns A. MTOCHMM'HS A. dumlccallis A. /llwescens A. viscosus A. oiscosus N RRL 13-1258 Lochhcad and N RRL B-2883 Lochhead Lochhead N RRL 13-1973 NRRL 13-1797 13-2882 Burton (1953) (1958) (1958) Gelatin liquefaction slow Starch hydrolysis... H28 production Chromogenesis yellow yellow brown (on Y. pa.) cream-pale black. brown.

Catalqqo Motility Citrate utilization N11 NO utilized as sole N source.

13 thiamin, or terregens factor (Lochhead 1953) needed for growth.

tcrregcns factor.

terregens factor -I- thiamine biotin.

TABLE IL-COMPARISONS BETWEEN KNOWN STRAINS OF ARTHROBACTER AND NEW STRAINS A. globiformis A. pasccns A. pascans A. simpler A. oer dam A. aurescem A. tumescem NRRL B-288O N RRL 1814 NRRL 13-2884 Jensen (1954) Sguros (1955) NRRL 13-2879 NRRL B-2881 N02 irom N03" N2 from N: nu.

Growth viscous in liquid media--. no yes very no yes no no Growth at 37 slight poor Litmus milk clearing slow, clearing sl0W reduction slow clearing roclearing soft curd,

, alkaline. clearing. dnction, clearing.

alkaline.

A. citreus A. terrcgens A. atrocyzmeus A. duodecadis A. flavescans A. viscosus A. viscosus NRRL B-1258 Lochhead and NRRL 13-2883 Loehhead Lochhead N RRL B 1973 NRRL*B=1797 13-2882 Burton (1953) (1958) (1958) N02 and N03.

Growth viscous in liquid media.-- no no no no no very very.

Growth at 37 C slight.

Litmus milk no change... no change".-. soft curd, no change. no ehange reduction reduction clearing clearing, clearing, basic. slightly alslightly alkaline. kaline.

Having fully disclosed our invention, We claim:

A process for obtaining a polysaccharide consisting of glucose, galactose, mannuronic acid and O-acetyl moieties that are respectively present in the molar proportions of 1:1:1:5 comprising inoculating a sterilized fermentation medium present in a paddle equipped fermentor and having the following composition per 100 ml.

Grams Commercial corn sugar 3.0 Enzyme-hydrolyzed casein 0.25 MgSO -7H O 0.08 M11804 KH PO 0.4

Water, q.s. a.d., 100 ml.

mainder of the fermentation, diluting the viscous medium With dilute aqueous methanol, removing unwanted cells and debris, adding ethanol and a salt selected from the group consisting of potassium chloride and potassium acetate to precipitate the polysaccharide, and isolating the precipitated polysaccharide.

References Cited by the Examiner UNITED STATES PATENTS 2,961,378 11/1960 Benedict et a1 31 3,000,790 9/ 1961 Jeanes et a1 19531 3,020,206 2/ 1962 Patton et al 19531 3,096,293 7/1963 Jeanes et al. 252316 OTHER REFERENCES United States Department of Agricultural Research Service CA-N-2l| pp. 1-3 and attached page bearing FIGS. 1-3. May 1962.

A. LOUIS MONACELL, Primary Examiner.

A. E. TANENI-IOLTZ, Assistant Examiner. 

